Chromosomal Location of Genes Differentially Expressed in Tumor Cells Surviving High-Dose X-ray Irradiation: A Preliminary Study on Radio-Fragile Sites

Altered gene expression is a common feature of tumor cells after irradiation. Our previous study showed that this phenomenon is not only an acute response to cytotoxic stress, instead, it was persistently detected in tumor cells that survived 10 Gy irradiation (IR cells). The current understanding is that DNA double-strand breaks (DSBs) are recognized by the phosphorylation of histone H2AX (H2AX) and triggers the ataxia-telangiectasia mutated (ATM) protein or the ATM- and Rad3-related (ATR) pathway, which activate or inactivate the DNA repair or apoptotic or senescence related molecules and causes the expression of genes in many instances. However, because changes in gene expression persist after passaging in IR cells, it may be due to the different pathways from these transient intracellular signaling pathways caused by DSBs. We performed microarray analysis of 30,000 genes in radiation-surviving cells (H1299-IR and MCF7-IR) and found an interesting relation between altered genes and their chromosomal loci. These loci formed a cluster on the chromosome, especially on 1q21 and 6p21-p22 in both irradiated cell lines. These chromosome sites might be regarded as “radio-fragile” sites.


Introduction
X-rays are widely used in various fields, especially in medicine for diagnostic and therapeutic purposes. There have been tremendous advances in these technologies, and the applications of radiation in medicine continue to increase. At the same time, there has been increased focus on the importance of understanding the effects of radiation on the human body, such as the mechanisms underlying radiation-induced DNA double-strand breaks (DSBs), radiation-induced signal transduction, and/or alterations in gene expression, which have been well studied [1][2][3]. On the other hand, the emergence of tolerant tumor cells during or after radiotherapy remains problematic, and the characteristics of these tumor cells remain unclear.
Genomic and chromosomal instability resulting from various mutational processes occurring within the genome and chromosomes leads to alteration in the expression of tumor-suppressor genes and is associated with tumor progression and tumor malignancy [4]. Radiation also triggers genomic changes as well as changes in gene expression and cellular characteristics [1,[5][6][7][8]. Muradyan et al. reported that high-dose X-rays induced genomic changes [9]. Some forms of genomic and chromosomal instability result in common genomic or chromosomal fragile sites [9]. Sarni et al. reported that there are common fragile sites that are susceptible to replication stress and are hotspots for chromosomal instability in cancer [10].
On the other hand, the number of radiation-induced DNA DSBs correlates well with probabilistic models of energy transfer to DNA by secondary electrons derived from ionizing radiation [1,11,12]. Therefore, we hypothesized the possibility of the presence of the common fragile chromosomal sites that are easy targets for radiation, and these may lead the radio-tolerant tumor cells during or after radiotherapy. In our previous study, we established a subclone of the p53-null non-small cell lung cancer cell line H1299 (H1299-IR) that survived 6 MV of 10 Gy X-rays and its increased cellular motility, invasiveness, and adhesion and the change of the gene expression were reported [7,8]. However, the mechanism how IR cells obtain their changes of the characteristics, and the gene expression has been unknown. There are several possible reasons for them: for example, IR cells may possess the genotype that can survive X-ray irradiation; they may be cancer stem cells; the accumulation of the DNA repair error may have occurred after DSB or SSB induction; and so on. As for the changes of the gene expression in IR cells, it is interpreted that different pathways from the DSBs based signal transduction may have arisen because the changes persist after passaging the cells. Among the abundant information on the changes of the gene expression obtained by microarray analysis, the present study focused on the relation between changes of the gene expression and their chromosomal locations.
In the present study, we preliminarily searched for fragile sites on tumor cells that survived 10 Gy X-ray irradiation by gene locus mapping via microarray analysis using H1299-IR and MCF7-IR cells.

Cell Culture
The human non-small cell lung cancer cell line H1299 and the human breast cancer cell line MCF7 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and Health Science Research Resources Bank (JCRB, Osaka, Japan), respectively. Both lines were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan) at 37 • C in a humidified atmosphere of 95% air and 5% CO 2 . To establish radiation-surviving cells, semi-confluent cell cultures were exposed to 10 Gy X-rays at room temperature using a linear accelerator (Mitsubishi Electric Co., Tokyo, Japan). The cells were immediately dispersed with 0.25% trypsin-EDTA (Sigma, St. Louis, MO, USA) and the cells were roughly spread onto culture dishes (100-mm diameter). After 14-30 days, all colonies were harvested and referred to as IR cells (i.e., H1299-IR and MCF7-IR).

Subcloning for MCF7-IR Cells
We subcloned radiation-surviving MCF-IR cells according to our previous report, where we established radiation-surviving H1299-IR cells [4]. Semi-confluent cultures were exposed to 10 Gy X-rays at room temperature using a linear accelerator ((MHCL-15S, Mitsubishi Electric Co., Tokyo, Japan). Cells were immediately dispersed with trypsin and spread onto Petri dishes (100-mm diameter). After 30 days, all colonies were harvested and established as MCF7-IR cell lines.

Colony Formation Assay
Cells that survived X-ray irradiation were analyzed using a clonogenic assay. The cells in logarithmic phase were irradiated with 6 MV of 10 Gy X-ray, and then they were immediately trypsinized and the viable number of the cells (10,000-100,000 cells) were seeded onto 60-mm dishes. The cells were cultured in DMEM containing 10% FBS and after 14-30 days, the cells were fixed with methanol and stained with 2% Giemsa solution (Kanto Chemical Co. Inc., Tokyo, Japan) to determine the number of colonies per dish. Values were corrected by comparison with the plating efficiency of the untreated cells. The viability was evaluated by dividing the number of colonies after irradiation by the number of colonies of unirradiated cells, which is given in percentage after the correction for plating efficiency.

DNA Microarray Analysis
The parental cells and IR cells were seeded onto 60-mm dishes and cultured for 24 h. Total RNA was extracted from semi-confluent cells using the QIAGEN RNeasy kit (Qiagen, Chatsworth, CA, USA) and was labeled and hybridized onto a human microarray chip that targeted approximately 30,000 human genes. The detected signals were then examined by computer analysis (Agilent Technologies Japan, Ltd., Tokyo, Japan).

Data Analysis
Among the 100 top genes having changed in the expression level more than 1.5-fold up-and down-regulations in microarray data, the information on the chromosomal location of genes was derived from two sources: National Center for Biotechnology Information (NCBI) and Basis Local Alignment Search Tool (BLAST). The relationship between the alteration of gene expression by irradiation and the chromosomal location was graphically indicated.

Irradiated Condition
The logarithmic phase of the cells cultured in the flask on a water-equivalent phantom with a 50-mm-thickness was irradiated with 6 MV therapeutic X-rays (MHCL-15S, Mitsubishi Electric Co., Tokyo, Japan) up to 10 Gy at a dose rate of 250 MU/min at room temperature.

Statistical Analysis
The statistics was analyzed using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). The comparison between two groups was performed by the Mann-Whitney's U test. The error bar indicates the standard deviation of the mean (±SD) of triplicate samples. Statistical significance was set at p < 0.05. All analyses were performed for duplicate experiments both in H1299 and MCF7.

Radio-Sensitivity of IR Cells
First, we compared all of the radiosensitivity of H1299, H1299-IR, MCF7, and MCF7-IR cells. Among the parental cell lines, MCF7 cells were >100-fold more sensitive than H1299 cells. Both types of IR cells were approximately 3.0-fold more sensitive than their corresponding parental cells ( Figure 1 and Table 1).

Functional Categories of Altered Genes
We categorized the functions of the altered genes located in chromosomes 1q21 and 6p21-22 (Tables 2 and 3). However, there were no common or related genes between H1299-IR and MCF7-IR cells for both up-regulated and down-regulated genes.

Functional Categories of Altered Genes
We categorized the functions of the altered genes located in chromosomes 1q21 and 6p21-22 (Tables 2 and 3). However, there were no common or related genes between H1299-IR and MCF7-IR cells for both up-regulated and down-regulated genes. Degradation of the antigenic proteins to peptides on MHC class II S100 calcium binding protein A3 (S100A3) [NM_002960] * S100 calcium binding protein A2 (S100A2) [NM_005978] Tumor suppressor function aquaporin 10 (AQP10) [NM_080429] Water-selective channel * Genes whose functional categories are unknown.

Discussion
In the present study, we found a clustered chromosomal region where gene expression was altered after high-dose X-ray irradiation (Figures 2 and 3). For up-regulated genes, these cluster regions are commonly seen in H1299-IR and MCF7-IR cells on chromosomes 1q21 and 6p21-22 (arrows in Figure 2). In contrast, the clusters in the common loci between H1299 and MCF7 were not shown in the down-regulated genes (Figure 3). Among the common chromosomal sites, 6p21-22 in H1299-IR cells is the location where up-regulated genes are particularly large in number (n = 18) (Figure 2). Similarly, the largest number of up-regulated genes in MCF7-IR cells was seen on 1q21 (n = 10). Note that 6p21 is a wellknown fragile site [13,14]. Such fragile sites have been studied mainly in carcinogenesis associating with tumor malignancies [15][16][17]. For example, 6q21 is commonly deleted in leukemias and primary breast cancer [16,17], and also contains the region for HECT domain and Ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) gene that under expressed in Wilm's tumor [18,19]. 6p21 is the largest region of the human genome that encodes about 230 genes in 3.6 megabases (Mb) of the histocompatibility complex (MHC). Therefore, physically, DNA would be easily attacked by irradiation in a large region of 6p21-22 and would cause the alternation of the tumor characteristics especially to the tumor malignancy.
In chromosome 1q21, 12 and 4 up-regulated genes were found in MCF7-IR and H1299-IR cells, respectively ( Table 2). The 1q21 region also plays an important role in the development and progression of tumors [20]. For example, in ovarian cancer and neuroblastoma, 1q21-22 is associated with a drug-resistant phenotype and a poorer response to chemotherapy [21,22]. Thus, it is a reasonable hypothesis that genes located at 1q21 might also affect radio-sensitivity.
Chromosomal fragile sites are defined as regions of the genome that exhibit gaps or breaks on metaphase chromosomes under conditions of partial replication stress [23], and it is particularly susceptible to DNA breakage [24]. To date, more than 100 fragile sites have been identified [25]. The common fragile site, FRA6F, is also located at 6q21 [25,26]. While, in the nuclei, loci in each chromosome are especially well ordered according to the chromosome territories (CTs) [27,28]. CT epigenetically affects global gene expression and regulation of the genome, including DNA methylation, remodeling, and other modifications [27,29]. There is one experiment that when cells that pulse are labeled with 3 H-thymidine, the labeling region of DNA is not labeled in smaller amounts on several chromosomes but is instead restricted to one of the chromosomes [27]. That means 3 Hthymidine interacts only with the limited region of the DNA. As the interaction between ionizing radiation and DNA occur stochastically depending on conditions such as dose, linear energy transfer, flux density, and oxygen conditions, the frequency of DNA damage depends on linear energy transfer to DNA and the number of secondary electrons and generated oxygen radicals [1,11,12]. These indicate the possibility that limited CTs may be attacked by ionizing radiation, the same as in the 3 H-thymidine labeling experiment.
According to the functions of the genes located on chromosome 1q21 and 6p21-22 listed in Tables 1 and 2, many genes encoding histone regulatory proteins were congregated at these loci in H1299 (Tables 2 and 3), and radiation-induced alteration of gene expression may affect some activities of histone-associated proteins. Also, as some tumor-suppressor associating genes and the genes associating with cellular progression, apoptosis, and the immune system were located in 1q21 and 6p21-22, these chromosomal loci might not only be physically fragile but have the important role for tumor characterization. The possibility that chromosomes 1q21 and 6p21-22 are fragile (stochastically susceptible to attack by ionizing radiation) and the molecular mechanisms of the semi-permanent persistence of the altered gene expression in the IR cells needs to be verified by repeated experiments with the various types of cells in the future.
In the present report, we have indicated, for the first time, the possibility of ionizing radiation-sensitive DNA fragile sites. These might affect DNA damage, change of temporary or continuous gene expression, and genomic instability ( Figure 4). Further study will be necessary to clarify the potential influence of ionizing radiation on gene expression by repeating the experiments with more samples. manent persistence of the altered gene expression in the IR cells needs to be verified by repeated experiments with the various types of cells in the future.
In the present report, we have indicated, for the first time, the possibility of ionizing radiation-sensitive DNA fragile sites. These might affect DNA damage, change of temporary or continuous gene expression, and genomic instability (Figure 4). Further study will be necessary to clarify the potential influence of ionizing radiation on gene expression by repeating the experiments with more samples.

Conclusions
We found a relation between genes with altered expression and their chromosomal loci, especially on 1q21 and 6p21-22, in cells that survived high-dose X-ray irradiation. These chromosome loci might be regarded to be "radio-fragile" sites.

Conclusions
We found a relation between genes with altered expression and their chromosomal loci, especially on 1q21 and 6p21-22, in cells that survived high-dose X-ray irradiation. These chromosome loci might be regarded to be "radio-fragile" sites.

Data Availability Statement:
The data that support the findings of this study are not publicly available. However, data are available from the authors upon reasonable request.